The molybdenum cofactor (Moco) consists of a mononuclear molybdenum coordinated to the sulfur atoms of a unique dithiolene group which is part of a substituted pterin derivative termed molybdopterin. Enzymes containing this cofactor are found in all three phylogenetic kingdoms of life, and biosynthesis of the cofactor follows the same pathway in prokaryotes and in eukaryotes including humans. Moco deficiency in humans is a severe inherited disease that manifests itself in neurological abnormalities and leads to premature death in he affected individuals. The first mutations in the genes encoding enzymes involved in Moco biosynthesis have recently been characterized from patients, and the effects of the mutations can be rationalized and analyzed using the structures of the E. coli proteins we have derived. In addition, the enzyme involved in dinucleotide formation in eubacteria might be a suitable candidate for the rational design of inhibitors that function as antimicrobial agents. The experiments outlined in this proposal will further advance our understanding of the enzymes involved in this conserved pathway through a combination of structural and biochemical experiments. Furthermore, studies of two proteins involved in the sulfur incorporation step during Moco biosynthesis provide important insights into the activation of ubiquitin and related protein modifiers (SUMO, NEDD8). The specific aims presented in this proposal can be summarized as follows: (1) Studies of the enzymes involved in the first step of this pathway. Specifically, we will determine the crystal structure of a MoaA protein, and further characterize the active site and function of MoaC. (2) Biochemical and structural studies of the enzymes involved in the formation of the dithiolene group of Moco. We will study the interactions between MoaD and its binding partners MoeB and MoaE and characterize the active sites of molybdopterin synthase and MoeB. (3) Further characterization of the proteins involved in the metal incorporation step through biochemical and structural characterization of MogA and MoeA. (4) Studies of the prokaryoto-specific steps during Moco biosynthesis. We will study the mechanism of dinucleotide formation by MobA and determine the crystal structures of MobA proteins from pathogenic bacteria as a starting point for the rational design of compounds with antimicrobial activity.